1. Field of the Invention
The present invention relates to flow cytometry apparatus, and more particularly, concerns an apparatus for detecting light parameters of particles flowing in a continuous stream, such apparatus utilizing a laser or other light source to direct a beam of light at the flowing particles.
2. Description of the Prior Art
Flow analysis of particles has been employed in the determination of various characteristics of individual particles. Flow cytometry devices have long been utilized for this purpose. In the broadest sense, a flow cytometry device as used and meant herein is a device which detects cells or particles as they flow, preferably individually, through an orifice. In addition to the capability of detecting particles flowing in a continuous stream, flow cytometry devices have been devised to determine volume, size, and other parameters of the flowing particles, particularly as such parameters are related to a source of light directed at them when flowing through the orifice.
In particular, many flow cytometry devices, including cell analyzers and cell sorters, rely upon a source of light energy directed against the flowing particles to thereafter establish certain measurements associated with the light which strikes the particles. For example, a device described in U.S. Pat. No. 3,710,933 measures cell volume through a Coulter-type orifice, and then measures light scatter and fluorescence of the particles that are being evaluated. In U.S. Pat. No. 3,826,364, an apparatus is disclosed which physically separates particles such as functionally different cell types. In this patented cell sorter, a laser provides illumination which is focused on the stream of particles by a suitable lens or lens system so that there is highly localized scatter from particles therein. In addition, a high intensity source of illumination is directed onto the stream of particles for the excitation of fluorescent particles contained therein. Luminescence from the excited fluorescent particles is then detected by suitable detection equipment. Certain fluorescent particles may be selectively charged and then separated by deflecting them into specific receptacles.
When utilizing lasers or other coherent light sources for illumination in flow cytometry device, obtaining optimum fluorescent pulse height resolution involves a balance between illumination uniformity, which determines the uniformity of fluorescence with particle position, and laser beam intensity which determines the available fluorescence photon flux. In presently known flow cytometry devices utilizing a laser for illumination, the laser beam focal waist is very narrow and thereby significantly affects sensitivity of the fluorescence signal. In circumstances where the fluorescence emitted from particles is weak, it is desired to have increased capability so that these weak fluorescent signals may be monitored. Accordingly, in the balance between illumination uniformity and laser beam intensity, as mentioned above, it remains a desirable feature to be able to improve the fluorescence sensitivity in the flow cytometry device.